Selective breeding of domesticated animals, based on phenotypic traits, has led to significant genetic improvement in a variety of breeds. For example, selective breeding techniques have resulted the production of diary cows which produce more milk and produce milk having improved fat and protein profiles. Reproductive technologies including artificial insemination and embryo transfer (i.e., transferring an embryo to an animal that did not produce it) have contributed to this genetic improvement. Advanced reproductive and molecular technologies, e.g., embryo cloning, and marker assisted selection (genetic screening), allow even greater control over the selection of desirable traits. However, these practices only allow selection and propagation of traits which are already in the gene pool of the species. In order to replace or modify existing genes or insert genes for traits which are not present in the gene pool of the species, a transgene must be introduced into the animal. Transgenes can be used to introduce disease resistance, alter the composition of animal derived products (milk, serum, etc.), produce pharmaceutical or nutriceuticals, or for other purposes.
A number of techniques can be used to create transgenic animals. Among the techniques which have been successfully used are: pronuclear injection of the transgene, nuclear transplantation, and injection of genetically altered stem cells into host embryos (chimera production).
Pronuclear injection is a commonly used procedure for germ line insertion of genes. While this technique is attractive because it can be used successfully with a range of animals, the inability to control integration of the transgene and the large number of ova which must be injected to obtain even a single transgenic offspring combine to make the technique rather inefficient, at least for animals other than mice. Moreover, animals which are deemed transgenic, e.g., by Southern analysis, may be mosaic, may contain the transgene but not express it, may express it in an undesirable manner, or may express correctly but fail to transmit it to offspring. Despite these difficulties, transgenic mice, sheep, goats, cattle and pigs have been produced using pronuclear injection.
Transgenic mice have been created by genetically manipulating murine embryonic stem cells (ESC), e.g., by injecting a transgene into the ESC, and then injecting the altered embryonic stem cells into a host embryo. The resulting mice are mosaics in which genetically altered cells contribute to a greater or lesser extent to the somatic and germ cells.
Nuclear transfer is a third approach to the generation of transgenic animals. In this technique the nucleus of a donor cell is introduced into a recipient oocyte. Offspring have also been reported in the bovine and sheep from cultured inner cell mass (ICM) cells and embryonic disks, respectively, using the technique of nuclear transfer.
Embryo transfer is a technique in which an embryo taken from a donor animal is transferred to a recipient animal who brings the embryo to term. Embryo transfer has successfully produced offspring when embryos from dwarf goats were transferred into standard goats Sugie et al., 1970.